Technical Abstract:
In vitro methods for gas production and fiber digestion were modified to measure gas production kinetics. Incubator and stirrers for the gas system were increased from 8 to 40 vials, and two sealed vials were monitored for changes in barometric pressure during fermentation. Samples(40-100 mg) were weighed into a 50 ml vial that had been weighed, calibrated for volume, and dcontained a small stirring bar. Media (0.7 ml) was added to each vial to wet the sample. Vials were connected to a manifold and continuously purged with CO2. Media (5 ml) was added to each vial in the water bath. Reducing solution (0.3 ml) was added to each vial which was disconnected from the manifold and lightly capped. Ruminal fluid (1200 ml) and solids (800 ml) were squeezed through 2 layers of cheesecloth. Squeezed solids (100 g) were blended with the 200 ml of media (previously chilled in ice, purged for 30 min with CO2, and reduced) for 45 sec. Strained rumen fluid (200 ml) was filtered through 4 layers of cheese cloth, the contents of the blender wer added and squeezed tightly. Inoculum was warmed to 39 deg C while being continuously stirred and purged with CO2. After resazurin indicator had cleared, 4 ml of inoculum was added while purging with CO2. Vials were capped with flanged butyl rubber stoppers, sealed with aluminum crimps, and immediately connected to the transducer by inserting the needle through the stopper. Vials were gently swirled after 6 h and every 24 h thereafter. Pressure was recorded every .01 h for the first .5 h, every .10 h for the next 6 h, and every .5 h until 96 h of fermentation. After fermentation was ended, media pH and residual DM and NDF were measured. Aspects of the procedure were evaluated and this procedure resulted in the highest reproducibility and maximum rates of gas production.